RCA (MouseBrain SNP)
This Protocol is modified based on
20240724_RCA-tissue-protocol.docx, featured with 1. low RCA primer
content; 2. activation step
1. fixation of the slides (～60min)
i. Incubate the slides in 200ul 0.1% GA in 4% PFA for 1**5-30 min**
Freshly prepare: 1µL 50% GA + 500 µL PFA
ii. Wash the slides in 200ul 1 x 2 min PBS-Tween 0.05%
iii. Wash the slides in 200ul 80% EtOH
iv. Assemble the PDMS chamber to glass slide
v. Wash the slides in 50 ul 1 x 2 min PBST
- Permeabilization of the tissue (~10min)
i. Incubate the slides in 0.04% pepsin in 0.1M HCl at 37C for 2 min (在四度冰箱)
ii. Wash the slides for 1 x 2 min with PBS-Tween 0.05%
3. Dehydration (~20min)
i. Incubate the slides in 80% ETOH for 10 min
ii. Incubate the slides in 100% ETOH for 2 min
iii. wash the slides for 3 x 2 min PBS-Tween 0.05% for rehydration.
4. Blocking (~30min)
| Reagents | stock | final | 1 x slide | 3 x slides |
|---|---|---|---|---|
| Rnase Free H₂O | ||||
| Ampligase Buffer | 10x | 1x | 10μl | |
| KCl | 1M | 0.05M | 5μl | |
| Formamide deionized | 100% | 20% | 20μl | |
| Probes oligo | 10μM | 0.05μM | 1μl | |
| BSA | 10μg/μl | 0.2μg/μl | 2μl | |
| RiboLock (Thermo) | 40U/μl | 1U/μl | 2.5μl | |
| tRNA (Ambion AM7119) | 10μg/μl | 0.2μg/μl | 2μl | |
| Total | 100μl |
i. Add the blocking solution into the chamber, mix by gentle pipetting (3-5 times) and
incubate the slides at room temperature (RT) for 30 min.
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5. Hybridization of Padlock probes (~3h)
| Reagents | final | Panel1 | (ul) | Panel2 | (ul) | Panel3 | (ul) |
|---|---|---|---|---|---|---|---|
| Rnase Free H₂O | |||||||
| Ampligase Buffer | 1x | 10x | 10x | 10x | |||
| KCl | 0.05M | 1M | 1M | 1M | |||
| Formamide deionized | 20% | 100% | 100% | 100% | |||
| Probes | 0.2μM | ||||||
| BSA | 0.2μg/μl | 10μg/μl | 10μg/μl | 10μg/μl | |||
| RiboLock (Thermo) | 1U/μl | 40U/μl | 40U/μl | 40U/μl | |||
| tRNA (Ambion AM7119) | 0.2μg/μl | 10μg/μl | 10μg/μl | 10μg/μl | |||
| Total |
i. Add the solution into the chamber, mix by gentle pipetting (3-5 times) and seal.
ii. Incubate the slides at 55oC for 18 min for denaturation and at 45o C for 120 min for
hybridization of the probes onto the target mRNA.
盖上盖子，烘箱温度可调成62-63和52-53
iii. Wash the slides for 3 x 10 min with washing buffer (10% formamide in 2X SSC) to remove unhybridized probes.
100µL 20X SSC + 100µL formamide + 800µL H₂O.
iv. Wash the slides for 3 x 1 min with PBS-Tween 0.05% to remove the remaining formamide, as it can deactivate the enzyme in the following step.
6. iLock probe activation (~1h)
| Reagents | stock | final | 1 x slide | 3 x slide |
|---|---|---|---|---|
| RNase Free H₂O | ||||
| Taq DNA Polymerase Buffer | 10X | 1X | 10 ul | |
| MgCl2 | 0.1 M | 8 mM | 8 ul | |
| BSA | 10 ug/ul | 0.2 ug/ul | 2 ul | |
| Taq DNA Polymerase (Thermo) | 5 U/ul | 0.1 U/ul | 2 ul | |
| RiboLock (Thermo) | 40 U/ul | 1 U/ul | 2.5 ul | |
| Total | 100 ul |
i. Add the solution into the chamber, mix by gentle pipetting (3-5 times) and seal.
ii. Incubate the slides at 51oC for 60 min for activation.
iii. Wash the slides for 2 x 1 min with PBS-Tween 0.05%.
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7. Ligation of Padlock probes (~2h)
The ligation of the hybridized padlock probes is mediated by SplintR ligase (PBCV-1 DNA Ligase) which can function with DNA:RNA hybrid molecules.
| Reagents | stock | final | 1 x slide | 3 x slide |
|---|---|---|---|---|
| RNase Free H₂O | 75.5 ul | |||
| SplintR Buffer | 10X | 1X | 10 ul | |
| BSA | 10 ug/ul | 0.2 ug/ul | 2 ul | |
| SplintR (NEB) | 25 U/ul | 2.5 U/ul | 10 ul | |
| RiboLock (Thermo) | 40 U/ul | 1 U/ul | 2.5 ul | |
| Total | 100 ul |
i. Add the solution into the chamber, mix by gentle pipetting (3-5 times) and seal
ii. Incubate the slides at 37℃ for 2 hours
iii. Wash the slides for 2 x 1 min with PBS-Tween 0.05%.
8. Rolling circle amplification (RCA, ~3h-17h)
| Reagents | stock | final | 1 x slide | 3 x slide |
|---|---|---|---|---|
| RNase Free H₂O | ||||
| Phi29 (Thermo) | 10 U/ul | 0.25 U/ul | 2.5ul | |
| Phi29 Buffer | 10X | 1X | 10 ul | |
| Glycerol | 100% | 0.2 ug/ul | 10 ul | |
| dNTPs | 10 mM | 0.25 mM | 2.5 ul | |
| NH2-dUTP | 2 mM | 0.05 mM | 2.5 ul | |
| BSA | 10 ug/ul | 0.2 ug/ul | 2 ul | |
| RCA Primer 1+2 | 10uM for both | 0.015uM for both | 0.15 + 0.15 ul | |
| Total | 100 ul |
i. Add the solution into the chamber, mix by gentle pipetting (3-5 times) and seal
ii. Incubate the slides at 30o C for 12-16 hours (For 2h, need to incubate at 37C)
iii. Wash the slides for 2 x 1 min with PBS-Tween 0.05%.
iv. Add 0.2% PEG9 in PBST mix by gentle pipetting (3-5 times) and incubate for 15-30 min at RT to fix the RCA products on the tissue.
Freshly prepare: 2µL PEG9 BS + 98µL PBST
v. Wash the slides for 3 x 1 min with PBS-Tween 0.05%.
vi. Wash the slides for 3 x 2-10 min with 65% formamide at 30o C on a PCR machine block or an Eppendorf Thermostat.
650 µL Formamide + 350 µL H₂O
vii. Wash the slides for 2 x 1 min with PBS-Tween 0.05%